JPH022368A - Production of fnudi restriction endonuclease and methylase - Google Patents

Abstract

NEW MATERIAL: A cloning vector containing FnuDI restriction endonuclease gene.

USE: A recombined gene for producing an enzyme for cleaving a DNA molecule to form a corresponding accurate fragment.

PREPARATION: This clone containing FnuDI restriction endonuclease gene is obtained e.g. by purifying a DNA originated from Fusobacterium nncleatum, digesting the DNA with a proper restriction endonuclease, ligating the obtained DNA fragment to a vector such as a plasmid, transforming a host cell such as Escherichia coli with the formed DNA mixture, digesting the formed library with FnuDI, transforming a host cell with the digested pool and subsequently selecting a clone containing the modified clone. A host cell containing the clone is cultured, and the product is subsequently recovered to obtain the FnuDI restriction endonuclease.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention The present invention relates to clones of the restriction endonuclease FnuDI and its modified methylases and methods of producing these enzymes from the clones.

Restriction endonucleases are a group of enzymes found in natural bacteria. Once purified from other contaminating bacterial components, restriction endonucleases can be used in the laboratory to cut DNA molecules into their respective precise fragments. Because of this property, each DNA molecule can be uniquely identified and its constituent genes can be isolated by fractionation. Restriction endonucleases have proven to be essential tools in modern genetic research. These enzymes are the biochemical "scissors" by which genetic engineering and analysis are accomplished.

Restriction endonucleases act by recognizing and binding to specific nucleotide sequences (so-called "recognition sequences") on DNA molecules.

When these enzymes bind to a DNA molecule, they cleave the molecule within or at one end of the sequence. Different restriction endonucleases have affinities for different recognition sequences. More than a hundred different restriction endonucleases have been identified in the hundreds of bacterial species studied to date.

Normally bacteria have only a small number of restriction endonucleases every second. These endonucleases are named after the bacteria from which they are derived. for example,
Haemophilus aegyptius is H3
Three different restriction endonucleases are synthesized, called O-E, Hae■ and )-1ae■. These enzymes are (AT)GGCC(＾■) and PuGCGC, respectively.
It recognizes and cleaves the sequences Py and GGCC. On the other hand, Escherichia coli RY1
3 only synthesizes one type of enzyme, EC0R1, and this enzyme recognizes the sequence GAATTC.

Without wishing to be bound by theory, it is believed that in nature, Lury-limited endonucleases play a protective role in the reproduction of bacterial cells. Thanks to these enzymes,
Bacteria are enabled to resist infection by foreign DNA molecules, such as viruses and plasmids, which, if left unchecked, would destroy or parasitize these bacteria.

Restriction endonucleases confer resistance to bacteria by binding to invading DNA molecules and cleaving them each time they encounter a recognition sequence. As a result of the destruction that occurs, many of the invading genes become ineffective and their DNA becomes fragmented by exonucleases.

The second component of the bacterial defense system is a modification methylase. These enzymes are complementary to restriction endonucleases,
This provides a means by which bacteria can protect and distinguish their own DNA from foreign infectious DNA. Modifying methylases recognize and bind to the same nucleotide recognition sequence as their corresponding restriction endonucleases, but instead of breaking this DNA, they chemically modify any of the nucleotides within that sequence by adding a methyl group. do. When this methylation occurs, the recognition sequence is no longer bound to or cleaved by the restriction endonuclease. The DNA of bacterial cells, vesicles, is always fully modified thanks to the activity of their modifying methylases and is therefore completely insensitive to the presence of their own endogenous restriction endonucleases. Unmodified DNA is susceptible to recognition and attack by restriction endonucleases
, DNA that can therefore be confirmed to be foreign
Only.

With the advent of genetic engineering techniques, it is now possible to clone genes and produce the proteins and enzymes they code for in greater quantities than are available using traditional purification techniques. The key to isolating a clone of a restriction endonuclease gene is that if the frequency of occurrence of such "]-nes is as low as 10-3 to 10', complex
The objective of the present invention is to develop a simple and reliable method for identifying clones of interest within a population of clones obtained using a "library" or "shotgun" method.

Preferably, the method should be selective so that the majority of undesired clones are destroyed, while the rare desired clones survive.

■ Type restriction - Modification systems are being cloned and their number is gradually increasing. The first cloned system used infection with bacteriophages as a means to identify or select restriction endonuclease clones [Hha II Hannet et al., Gen.
e, 3: 97-112 (1978): Ec
oRU: Niosykh et al, Ho1e
c, gen, Genet, 178
: 717-719 (1980): PstI:
Walder et al., Proc., Nat.

8cad, Sci, USA, 78.1503-1
507 (1981)], does the existence of a restriction-modification system in bacteria cause bacteria to become bacteriophores? In principle, cells carrying the cloned restriction-modified genes can be selectively isolated as strains that emerge (survive) from the phage-infected library because they are able to resist infection by phages. can do. However, this method proved to be of limited value. Specifically, it has been found that cloned restriction-modification genes do not always express sufficient phage resistance to allow selective survival.

Another cloning method incorporates the originally plasmid-derived system into the l:, C01i cloning plasmid [EcoRV: Bouauel
Nucleic Ac
1ds Res, 12:3659-3676 (19
84); PaeR7: Ginaeras and
Brooks.

Proc, Natl, Acad, Sci,
USA, 80:402-406 (1983):
Theriault and Roy, Gene;
19:355-359 (1982): Pvuff
: Blumenthal et at,
J, Bacterio1164: 501-509
(1985)].

A third method, one used for cloning many systems, is described in our patent application no. 70707.
Select for active methylase genes related to No. 9 [BsuRI: Niss et al., Nucl.
eic^cids Res, 13: 6403-
6421 (1985)]. Because restriction and modification genes tend to be closely linked, clones containing genes from both regions can often be isolated by selecting for only one gene. However, selection based on methylation activity does not always result in a complete restriction-modification system; on the contrary, only methylase genes may be obtained.URspRI: Szomolanyi et a
Gene, 10:219-225 (1980
); BCnI: Janulaitis et al.
,,Gene20:197-204 (1982):
B suRI: K15s and Bald
auf.

Gene, 21:111-119 (1983): and MspI: Walderet at, J.
, Biol, Chem, 258:1235-1241
(1983)].

A potential obstacle to cloning restriction-modification genes is #ll! The aim is to introduce the endonuclease gene into a host that is not protected by ili. If the methylase and endonuclease genes are introduced together as a single clone, the methylase should embellish and protect the host DNA before the endonuclease has a chance to cleave it. Therefore, in some cases these genes are ordered (i.e. methylase first, then endonuclease)
Cloning may only be possible in

Another obstacle to cloning restriction-modification systems is that
This is due to the discovery that some Seidan strains show the opposite reaction to cytosine modification. Indeed, such strains have a system for destroying DNA containing methylated cytosines [Raleigh and
Wilson, Proc, Natl, AcadS
ci, USA, 83: 9070-9074 (1
986)]. In these strains, the cytosine-specific methylase gene cannot be easily cloned either on its own gene or together with its corresponding endonuclease gene. To avoid this problem, E. coli mutant strains lacking these systems (M
CrA and MCrB) must be used.

Purified restriction endonucleases, and to a lesser extent modified methylases, are useful tools for characterizing and rearranging DNA in the laboratory, and the bacteria that synthesize these enzymes are essential. Obtaining strains by recombinant DNA technology has commercial appeal. Such strains, if obtained, would be extremely useful, since they would not only provide a means of producing them in commercially useful quantities, but would also simplify purification operations. Become.

SUMMARY OF THE INVENTION According to the present invention, Fusobacterium nu
Clones containing genes for the FnlJDI restriction endonuclease and modified methylase from S. cleatum D are provided, as well as methods for producing these enzymes.

More particularly, the present invention relates to clones expressing the restriction endonuclease FnuDI, an enzyme that recognizes the DNA sequence GGCC and cleaves it between G and C. [
ul, Δ, C, P,, HcBride B, C, Vo
vis G.

F, and Sm1th H, Nucleic A
c1ds Res, 6: 1-15 (1979)
Reference, the disclosure of which is hereby incorporated by reference.

The FnuDI restriction endonuclease produced according to the present invention is a combination of FnuDIr and FnuDIIl of the restriction endonucleases mixed in F, nucleatuIllD.
- does not include.

A preferred method of cloning this F3 cord is F.
nucleatum D, isolated clones containing DNA encoding the FnuDI modified methylase, and screened them to determine whether they also contained the FnuDI limited endonuclease gene. It consists of identifying clones.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to clones of the FnuDI restriction and modification gene and the restriction endonuclease FnllDI produced by such genes.

These FnuDI genes are cloned by a method that takes advantage of the fact that certain clones selected on the basis of containing and expressing the FntlDI modified methylase gene also contain the FnuDI restriction gene. Such a loan DNA, tFnuDI
Resistant to digestion by restriction endonucleases and Haenl restriction endonuclease. This resistance to digestion provides a means to selectively isolate clones encoding FnuDI methylase and restriction endonucleases.

A preferred method of the invention for cloning and expressing the FnuDI restriction gene and methylase gene is shown in FIG. 1 and includes the following steps.

(1) Fusobacterium nucl
Purify the DNA of Eatum D. F, nuc+
eatum D is Lui et al,, 5upr
Dr. Hichael Smlt
h, Dept, ofBiochemistry,
Faculty or Medicine T
heUniversity of Br1tish
Columbia, VancouverCanada
Provided by.

(2) Digest this DNA with a restriction endonuclease such as @indm.

(3) Digest the digested DNA with one or more FntlDI
pB R322 (ATCC 37017) containing the site
Connect to the O-ning vector as follows. connected D
The NA is transformed into a suitable host such as Escherichia coli strain R11 (ATCC 31343).

(4) Inoculate the transformed mixture into Amp plates containing antibiotics such as ampicillin to select transformed cells. After medium N, the transformant colonies are collected together to form a cell library.

(5) Completely purify all recombinant plasmids from this cell library to create a plasmid library.

(6) Transfer this plasmid library to FnuDI
Restriction endonuclease, or Haemophil
+-+aem derived from ushaemolyticus
Digest completely with an equivalent endonuclease such as FnuDI (or HaeII) digestion specifically destroys unmodified clones that do not contain methylase and increases the relative frequency of Fnu[)I methylase clones.

(7) Digested plasmid library with E.

transformed into a suitable host such as CO1+ Rltl,
Transformants are recovered by inoculation onto a selective medium. These colonies were collected and their DNA was converted into Fnu[)DingI! Analyze the presence of decorative genes. That is, the plasmid carried by the colony was purified, and FnuDI (or l-1a
eIff) Incubate with restriction endonuclease to determine if it is less resistant to digestion. In addition, the total DNA of the cell (chromosome and plasmid) is also
Incubate with FnuDI (or HaeI[I) restriction endonuclease.

The DNA of the FnLIDI modified S gene whole S gene clone should be sufficiently modified that both plasmid DNA and total DNA should be substantially resistant to digestion.

(8) Step (7) Prepare a cell extract of the identified FnuI)I methylase clone, and
Clones carrying FnLIDI restriction endonuclease C are identified by assaying uDI restriction endonuclease activity.

(9) FnuDI restriction endonuclease production of clones can be increased by increasing gene dosage using high copy number vectors and by increasing transcription rate using highly active exogenous promoters. can.

(10) 8 clones carrying the F nuD I restriction and modification gene! FnuDI restriction endonuclease can be produced by growth in an ampicillin-containing enriched medium in a fermentor.

Cells are collected by centrifugation, disrupted by sonication, and FnLI
A crude cell extract containing DI restriction endonuclease activity is produced.

(11) The crude cell extract containing F nuD I restriction endonuclease activity is purified by standard protein purification techniques such as affinity chromatography or ion exchange chromatography.

Although the procedure outlined above is a preferred embodiment of the invention, it will be apparent to those skilled in the art that the procedure described above may be modified by techniques known in the art.

The present invention will now be illustrated by giving specific examples that are currently preferred; however, these examples are merely illustrative, and the present invention is not limited to these examples unless specified in the claims. I want to be considered something.

Example Cloning of FnuDI restriction endonuclease gene (1) Purification of DNA: Frozen Fusobacterium nuc+ea
10 J of tum D cells were thawed on ice for 1 hour.
0d 25% sucrose, 50mHTris pl+
8.0. 10m1 of 0.25H[[
l0IQ/Inl of lTΔpH8,0Il13 and 6d
Lysozyme (in 0,25HTriS DH8,0) was added. After keeping this suspension on ice for 2 hours, one of the 24 precepts was prepared.
%TritOnX-100,501118 ris p
H8,0,67mHEDTA and 5ml 10% SD
S was added to lyse the bacteria. The resulting solution (previously 0
．． Extracted with 60 ml of phenol (70d) and di[10] form (equilibrated with 58 Tris, pH 8.0).

The obtained emulsion was centrifuged at 10 Kppm for 30 minutes 1) to separate the phases. The viscous upper phase was transferred to a new bottle and extracted once again with phenol and chloroform.

After centrifuging the obtained emulsion again, the F phase was diluted with DNA buffer (10mHTrisptl 8.0.1mHED).
This buffer was dialyzed against TA) with four changes of buffer. The dialyzed solution was then digested with RNaSe at a final concentration of 200 μg/d for 1 hour at 37°C, 58 NaCρ was added to a final concentration of 0.48, and 0.55 volumes of isopropyl alcohol were added to precipitate the DNA. I let it happen. The precipitated DNA was taken out by winding it around a glass rod, dried, and then dissolved in a DNA buffer solution at a concentration of about 300 s/d and stored at 4°C.

(2) DNA digestion: F, nucleatum D DNA 30/4
7 in 300μ restriction endonuclease digestion buffer (10mHTris 0117.5.10mHHaG!
2.10mH Melt Ethanol.

50mHNaC1! ). )(ind m
60 units of restriction endonuclease were added and the resulting solution was incubated at 37° C. for 2 hours. Digestion was stopped by heating to 72°C for 12 minutes.

(3) Ligation and transformation: F digested with Hind m, nucleatum D
pBR322 (ATCC37017) 3, which was obtained by cleaving 60μ of DNA with Hind m and dephosphorylating it.
Mixed with Iig (301j1).

10x ligation buffer (500mHTris pit
7.5.100+nHH(ICi!2.100mHDT
T, 5fflHATP) was added at 201ii, and 907J of sterile distilled water was added to the colander to make the volume 200J.

7.5 μ of T4 DNA ligase was added and the resulting solution was incubated at 17° C. for 4 hours. This solution was sterilized by extraction with 20 mL of chloroform, and then briefly centrifuged for 15 seconds to clarify it.

SSC/CaG of 800111 with 100 composition of coupling solution! 2
(50mHNaQ!

5m)l citrate Ha, 67fflHCaQ!
2) and cooled on ice, coli RRl (AT
CC31343) D. pitent cells were added for 1.7 d. The resulting solution was incubated at 44°C for 4 minutes, then 10 d of Luria-broth (L-broth) was added and incubation was continued at 37°C for 3 hours.

(4) The cell library-transformed culture was gently centrifuged, the supernatant was discarded, and the cells were resuspended in 1-broth for about 1.2 days.

Approximately 200 of the resuspended cells were treated with 1 ml of ampicillin.
8 pieces of turta-agar (L) containing 100 JJ/m
-aaar) plate. The plate was incubated overnight at 37°C. In order to collect all the transformed cells that grew on the surface of the plate, each plate was treated with 2.5-10 mH Tris pH 7.5,
Fill with 10 mHHaC12, scrape off all colonies, and bouquet the resulting suspension into one tube.

(5) Add 1 plasmid library and 2.0 d of cell library to 100 ml of ampicillin.
Contains 咒/d-broth 500mf! inoculated inside.

After culturing with 11cI overnight at 37°C, the cells were centrifuged at 4K rpm for 5 minutes. Discard the supernatant and remove the cells by 25% of the IM.
Sucrose, 50m)4 was resuspended in Tris pH 8.0 at room temperature.

0.25HEDTA, DH8,0 5m, J3J:
and 101119/dlJ Sozyme (0,25HTris
, pH 8.0) were added.

After leaving the resulting solution on ice for 1 hour, 1% of 12 m
Triton X-100, 50mHTris pH
8,067 mHEDTA was added and the suspension was gently swirled to induce cell lysis.

Transfer the lysed mixture to a Sod tube at 17Kr.
l)m, centrifuged for 45 min at 4°C. The supernatant was removed with a pipette. Weigh 20.0 g of solid CsC1 and place it in a 50 d plastic screw cap tube.
22.0 g of the 1st suspension was put into this tube with a pipette and mixed. 5 ■/d of ethidium bromide (10 mHT
ris pH8,0,100mHNap! , 1mH
(in EDTA) was added +, 0 precepts. Add this solution to 5/8
1nx3in centrifuge'? Move to 22 books, BeCkma
n Ti 70 rotor at 44K rpm.

Rotate for 42 hours at 17°C. To collect the plasmids, these duplexes were listened to, illuminated with ultraviolet light, and the lower two fluorescent bands were collected with a syringe. The lower band 19 from each duplex was combined and the ethidium bromide was removed by extraction four times with equal volumes of water-saturated, water-cooled n-butanol.

The extracted solution was dialyzed against DNA buffer with four changes and then diluted with 2 volumes of isopropanol and enough 5HNaCl to give a final concentration of 0.4M.
! was added to precipitate the nucleic acids. The resulting solution was left overnight.
ppm in v&15 stored at 20°C and centrifuged at 0°C for 15 minutes. After discarding the supernatant and air-drying the pellet for 15 minutes,
00pi's 10+nHTris Dll 7.5.1m
It was dissolved in HEDTA and stored at -20°C. The concentration of plasmid DNA was approximately 100 Mg/d.

(6) Digestion of plasmid library: 6i (60
The plasmid library of m) was diluted in 450ml of restriction endonuclease digestion buffer (Section 2 above).

60 units (7.5 m ) of Haell restriction endonuclease were added and the tubes were incubated at 37°C for 1 hour, then heated to 72°C for 12 minutes to stop the reaction.

(7) Transformation: 12.5 d (0.18/l) of the digested library was subjected to SSC/CaG! at 100 degrees. 2 (h2 term 3) J
Water-cooled competent E. coli at 3 and 200 degrees.
Mixed with Rltl. Stir the resulting mixture for 3 minutes at 42
After warming to ℃, L containing 100 g/d of ampicillin
- inoculated onto agar plates. This plate overnight 3
Incubated at 7°C. The number of transformants was reduced by about 103 times by HaeII digestion. This l-1a
From those that survived the ELL digestion, 14 colonies were collected and each was treated with 1-
Minicultures were prepared by inoculating into broth and master stocks were prepared by streaking onto L-agar plates containing ampicillin.

(8) Analysis of surviving individuals: The 14 surviving colonies described in Section 7 above were
0nf! The plasmids they carry were grown as described by Birnboin and Doly, N.
UCLEIC Acids Res, 7: 151
The following m1nipr applying the method of 3 (1979)
Prepared by ep purification method.

m1niprep method: Each culture was centrifuged at 8 rpm for 5 minutes, the supernatant was discarded, and the resulting cell pellet was incubated with 25 mHTris, 1,0, and 10 mHE containing IIIFg/rd lysozyme.
Resuspended in DTA, 50 mH glucose, pH 8.0. After 10 minutes at room temperature, add 2.
(0.2HNaO!11χ SDS of lnf! was added and the tubes were shaken to lyse the cells and then placed on ice. After the solution became clear, each 3M sodium acetate (p
H4,8) was added 1.5 d each and shaken. The resulting precipitate was centrifuged at 15K rpm and 4°C for 10 minutes to settle. The obtained supernatants were each poured into a centrifuge tube containing three volumes of isopropanol and mixed. After 10 minutes at room temperature, the centrifuge tube was centrifuged at 15K rpm for 10 minutes to pellet the precipitated nucleic acids.

The supernatant was discarded and the pellet was air-dried for 30 minutes at room temperature.

After drying, the pellet was coated with 850 ml of 10mHTris,
Resuspend in 1 m HEDTA, pl+ 8.0.

58 NaC1! The resulting solution was added to Eppen containing 575 pieces of isopropanol.
The mixture was transferred to an orf duplex and precipitated again at room temperature for 10 minutes. The tube was then spun in the microfuge for 45 seconds, the supernatant was discarded, and the pellet was air-dried. This pellet was then treated with RNaSe at 1ooIJ3/ml
Containing 5001J1 10mH 1mHEDT
RNA was digested by dissolving in pl+ 8.0 and incubating for 1 hour at 37°C. 50 years old 5HNap!
, then add 350% isopropanol to remove DNA.
was precipitated again. After 10 minutes at room temperature, the DNA was precipitated by centrifugation for 45 seconds, the supernatant was discarded, and the resulting belene was
0 scraps of 10mHTris, 1mHEDT 8p to 8.0
redissolved in. Then plasmid m1niprep
(small preparation) was digested with FnLIDI and Hind [[l] and analyzed.

(9) F nuD I medylase gene clone:
Eight of the 14 plasmids analyzed were sensitive to Hae F, nuc+eatum D
They were found to carry various fragments of DNA. These plasmids were discarded. The remaining six plasmids are resistant to HaelIliI conjugation;
It was found that they commonly carried a 5.5 Kb Hind m fragment. These plasmids, typified by pFnuDIRH2-33, appeared to be identical.

Several of these were selected and shown to encode not only FnUDI modification methylases, but also FnuDI restriction endonucleases.

This sample is the AmericanType C
U1ture Co11ection 8TCC405
It has been deposited as No. 21.

(10) F nuD I restriction gene clone: pF
nuDIRH2-33 and similar plasmids are isolated from E. coli RRI carrying these plasmids.
was found to encode and express the FnuDI restriction endonuclease. 50 trtl of culture of this clone was added at 100 m/I.
dL,-broth containing ampicillin at 37°C overnight. The cultures were centrifuged for 5 minutes in RPR5, and the resulting cell pellets were each diluted with 3 ml of cell lysis buffer (10 mHTrlSl) H7, 5, 10111.
8 mercaptoethanol, resuspended in 0.1 m HEDTA. A solution containing 10 Rg/trdl of lysodeme in the same buffer was added in 0.5 ml portions, and the resulting suspension was left on ice for 2 hours. This suspension was heated to -20°C.
After freezing overnight, thaw on ice and add 0.005X Tri
3. Cell lysis buffer containing tOn X-100.
Cell lysis was induced by vigorous mixing for 5 d. The lysed solution was briefly centrifuged for 5 minutes to solidify the MM, and the endonuclease activity of the supernatant was assayed as follows.

Thirty-five micrograms of purified λ phage DNA was diluted in 700ml restriction endonuclease digestion buffer (Section 2 above). Pour this solution into 6 tubes, the first one containing 1
100d each was dispensed into the remaining 5 bottles. 7.5 parts of extract were added to the first duplex to give 1 part of extract per 1 g of DNA. Next, remove 50 portions from the first tube and transfer to the second duplex.0.3111. /11
It was set as 9. Continuously, 50 samples were transferred to tubes 3, 4, and 5 in sequence, and tubes 3 (o, 1111/埒>, 4 (
0,03/J/#) and 5 (0,001 d/m). No extract was added to the 6-necked tube, which was used as a negative control. After incubating the tubes for 1 hour at 37° C., 20 ccg of sample from each tube was analyzed by gel electrophoresis. These extracts contain approximately 3X10'u of FnuDI per volume.
It was found that it contained a restriction endonuclease. This is approximately 1 x 106 cells;
It is equivalent to (Figure 3).

(11) 5.5Kb fragment 17) pUc19 (7
) Transfer: DN of purified pFnuDIRH2-33
A 5# (50 recommendations), 20 untt @ ind
I[[[[[[[[]]] was prepared in 100% restriction endonuclease digestion buffer (Section 2 above) containing restriction endonuclease. The solution was incubated at 31°C for 1 hour and then heated to 72°C for 12 minutes to stop the digestion. Digested pFnuDIRH2-33 DNA 3IJ3 (
60m) of Hind I [I cleaved with l and dephosphorylated] UC19 (ATCC 37254) (7,
5p1>. 10gf! The volume was brought to 100 by adding 10X ligation buffer (Section 3 above) and 22.5 μm sterile distilled water. 4 Cod T4 DNA ligase was added and the 1q solution was incubated at 17°C for 4 hours. This ligation was extracted with 15 ml of chloroform, sterilized, and clarified by gentle centrifugation.

Thus sterilized connection'#12.5IJJ! , , 10
0d CaC1!2/SSC (paragraph 3 above) and 20
0IIi (7) Ice-cold competent E, coli
1111. The resulting mixture was incubated at 42°C for 3 min, then treated with ampicillin-containing L-agar.
inoculated onto plates.

The plate was incubated overnight at 37°C.

2.5d of 10mHTrys C87 on this plate,
Transformants were collected by filling 510 mHH (IQ! 2 and scraping the colonies together to form a pool. This pool was inoculated into 5001 nf! ampicillin-containing 1-broth, and the plasmid preparation was purified ( (Section 5 above). Five volumes of the purified plasmid were
on restriction endonuclease buffer (section 6 above)
Digested with 40 u of Hael[I restriction endonuclease. 12.5μ of the digested DNA was
, col+ RRI (Section 7 above), and the surviving transformants were transformed with ampicillin-containing:6' 1-
Harvested by inoculating onto agar plates and incubating at -@31°C.

14 l17. ] was collected, and m1nipr
H
A plasmid constructed from 1) UCl3 with a 5.5 Kb fragment inserted into the ind m site was identified. 1
Three plasmids were found to possess this structure.

These carry the b fragment in 5.5 and are HacI[l
It showed complete resistance to digestion. The other two of these plasmids, pFnuDIRH102-1 and pF
E. coli R carrying nuDIRH102-4
Crude cell extracts of RI were assayed for Fnu[)I restriction endonuclease activity.

These extracts contain approximately 1 x 105 u per ml of extract.
nit (3 x 106 units per gram of cells) was found to contain FnuDI restriction endonuclease.

pFnuDIRH2-33 pFnuDIRH102-
1, coli RRl is Fnul) I
It can be used as a preferred host in purifying restriction endonucleases. This strain may be grown to stationary phase in ampicillin-containing L-broth in a fermenter at 37°C, then centrifuged to collect the cells, and immediately disrupted to prepare an extract, or kept at 70°C until use. It may be stored frozen.

[Brief explanation of the drawing]

FIG. 1 outlines the method for cloning and producing the FntJDI restriction endonuclease. Figure 2 shows Fnuc+eatum encoding the FnuDI restriction endonuclease and modified methylase.
This is a restriction map of the 5.5 Kb Hind m Vf+ fragment of DNA of D.
7017) ノ@ +ncj m site to construct pFnuDrRH2-33, and its 1m pu
C19 (ATCC 37254) into the Hindll[ site to create pFnuD[RH102-1]. Figure 3 shows F, c containing pFnuDIRH2-33.
Fnu[) in cell extracts of oliRRI (ATCC31343) ■Aga[) exhibiting restriction endonuclease activity
] - This is a photo of Sugeru. Hindllll FIG, 2 Hindllll IG3

Claims (14)

[Claims] (1) Cloning vector containing the FnuD I restriction endonuclease gene. (2) A transformed host containing the cloning vector according to claim 1. (3) FnuD I gene is ￥Fusobacter
The cloning vector according to claim 1, which is excised from iumnucleat-¥¥um¥D. (4) Cloning vector containing the FnuD I modified gene. (5) A transformed host containing the cloning vector according to claim 4. (6) (a) ¥F. (b) isolating clones containing the FnuD I modified gene; (c) screening clones containing the modified gene; (d) using FnuD I restriction endonuclease. A method for cloning the FnuD I restriction endonuclease gene, comprising isolating a clone containing the gene at the same time. (7) The library is (a) ¥F. DNA derived from nucleatum￥D
(b) digesting the purified DNA to form DNA fragments; (c) ligating the fragments into a cloning vector; (d) transforming host cells with the cloning vector of step (c). 7. The method of claim 6, wherein the method is formed by transforming the cell library to form a cell library; and (e) purifying a recombinant vector from the cell library to form a plasmid library. (8) The method according to claim 7, wherein the cloning vector is pBR322. (9) The host cell is ¥E. 8. The method according to claim 7, which is mcrB^- and hsdR^- strains of E. coli. (10) FnuD I modified genes were extracted by digesting the plasmid library with FnuD I to form a digested pool, transforming this digested pool into host cells, and selecting clones containing the modified genes. 8. The method according to claim 7, wherein a clone containing the peptide is isolated. (11) (a) ¥F. (b) digesting the purified DNA with a suitable restriction endonuclease to form a DNA fragment; (c) ligating this fragment into a cloning vector to form a DNA mixture. , (d) transforming host cells with the DNA mixture of step (c) to form a library; (e) isolating clones containing the FnuD I modified methylase gene; and (f) transforming the FnuD I modified methylase gene. (g) culturing host cells containing the clone of step (f); (h) extracting FnuD from this culture; A method for producing FnuD I restriction endonuclease, comprising recovering FnuD I restriction endonuclease. (12) The method according to claim 11, wherein the cloning vector is a plasmid or a viral DNA molecule. (13) Claim 12, wherein the plasmid is pBR322.
The method described in. (14) The method according to claim 12, wherein the plasmid is pUC19.